Calcium and cyclic AMP (cAMP) are important factors in the mechanism of episodic signaling in hypothalamic GnRH neurons. The observation that cAMP production in GT1-7 neurons is stimulated by increased extracellular Ca2+, and the Ca2+ channel agonist, BK-8644, and is diminished by low extracellular Ca2+ and treatment with dihydropyridine analogs, is consistent with activation and/or inhibition of the calcium-dependent adenylyl cyclase type I (ACI) expressed in these cells. These findings indicate that cAMP production in GnRH neuronal cells is maintained by Ca2+ entry through voltage-sensitive calcium channels, leading to activation of ACI, and that Ca2+ influx-dependent activation of ACI acts in conjunction with AC-regulatory G proteins to determine basal and agonist-stimulated levels of cAMP production. Also, cAMP-induced activation of cyclic nucleotide-gated channels (CNG), promotes Ca2+ entry and caused increase in GnRH secretion. The degree of coincidence between pulses of GnRH and cAMP under basal conditions was determined by concomitant measurements of both responses in samples collected from perifused GnRH neurons. We found that major GnRH pulses in basal GnRH release from perifused GT1-7 neurons frequently corresponded with the lowest cAMP level, indicating an inhibitory action of increased GnRH concentrations on cAMP production. Agonist activation of GnRH receptors leads to stimulation of phospholipase C, formation of IP3, mobilization of Ca2+ from intracellular stores, GnRH release, and cAMP production. Modulation of GnRH receptor activity in GnRH neurons by GnRH agonist and antagonist analogs and measurements of GnRH and cAMP production were used to investigate the regulatory mechanisms underlying GnRH and cAMP pulsatile release. The GnRH agonist D-Ala6Ag was used to evaluate cAMPs response to GnRH-R receptor activation. Continuous exposure of perifused GT1-7 neurons to high, GnRH receptor agonist analog D-Ala6Ag causes an increase in GnRH pulse amplitude and transiently stimulates cAMP production followed by prolonged inhibition. This is consistent with the findings that high GnRH concentrations cause coupling of GnRH-R to Gi and inhibited cAMP production. During the treatment of perifused GT1-7 neurons with low GnRH receptor agonist concentrations the amplitude of GnRH pulses remained unchanged, with concomitant increase in cAMP pulses. These findings indicate that at low GnRH concentrations GnRH receptors couple to adenylyl cyclase stimulatory G proteins and cause an increase in cAMP production. Thus, GnRH exerts biphasic effect on cAMP production where low GnRH concentrations stimulate cAMP production and at high concentrations GnRH inhibit cAMP production. Inactivation of GnRH receptors by both peptide and non-peptide GnRH antagonists abolishes pulsatile GnRH release. The cessation of pulsatile GnRH release is dose-dependent and switches to a monotonic increase at high GnRH antagonist concentrations. Cessation of pulsatile cAMP release was also evident during GnRH antagonist treatment, and cAMP production monotonically increased with rising GnRH antagonist concentrations, suggesting that GnRH/GnRH-R mediated inhibition is required to maintain both pulsatile GnRH and cAMP secretion. The GnRH antagonist-induced inhibition of GnRH-R, and an increase in cAMP production, may account cAMP-mediated increase in GnRH secretion. These data suggest that pulsatile cAMP secretion in GT1-7 neurons is driven by a GnRH/GnRH-R autoregulatory system, in which dose-dependent switching of GnRH-R coupling to adenylyl cyclase stimulatory (Gs) and inhibitory (Gi) G proteins mediates both pulsatile GnRH and cAMP secretion. The lack of expression of cAMP receptors in mammalian cells, as well as GT1-7 neurons, indicates that in contrast to invertebrates, where cAMP and its receptors provide for pulsatile cAMP release, in mammalian cells the pulsatile cAMP is differently regulated. Activation of PDE by PKA has been reported to have a role in establishing cAMP oscillations, which might constitute a biological clock for GnRH pulsatile release. Treatment of perifused GT1-7 neurons with the PKA inhibitor H-89 increased the pulse amplitude but not pulse frequency for both GnRH and cAMP. These data suggest that under basal conditions cAMP inhibitory adenylyl cyclase V and VI are tonically activated by PKA and mediate inhibitory effects on both GnRH and cAMP secretion. In summary, it is evident that pulsatile cAMP secretion in hypothalamic GnRH neurons is driven by a GnRH autoregulatory system in which changes in GnRH pulse amplitude cause an asynchronous relation between GnRH and cAMP pulses. The pulses synchronize after occurrence of the major GnRH pulse, providing a resetting mechanism by which GnRH and cAMP pulses may synchronize. Inhibition of pulsatile cAMP release by PDE inhibitors did not affect pulsatile GnRH release, suggesting that cAMP does not participate in the regulatory mechanism that is essential for GnRH pulsatility. Thus, it is evident that although cAMP is an active participant in GnRH secretion, it is not an essential component that drives pulsatile GnRH release, which is dependent on a GnRH autoregulatory system in hypothalamic GnRH neurons. Agonist activation of the neuronal GnRH receptor stimulates the phospholipase C InsP3/Ca2+ signaling pathway, increases or inhibits cAMP production in a dose-dependent manner, and modulates the frequency and amplitude of pulsatile GnRH release. G protein-coupled receptors also stimulate nitric oxide (NO)-sensitive soluble guanylyl cyclase (sGC) by increasing intracellular calcium (Ca2+i) and activating Ca2+-dependent nitric oxide synthetase (NOS). However, agonist activation of GnRH receptors increases Ca2+i but also causes dose-dependent inhibition of cGMP production. Such inhibition of cGMP production was GnRH receptor-specific, and was abolished by prior treatment with a GnRH-R antagonist. cGMP production increased in a dose-dependent manner during treatment with the calcium channel agonist Bay K 8644, indicating that the mode of increased Ca2+i determines its stimulatory action. The Bay K 8644-stimulated cGMP production was inhibited in a dose-dependent manner by GnRH, and treatment with the L-calcium channel blocker, nifedipine, did not potentiate the inhibitory effect of GnRH. Basal cGMP production was not Ca2+-dependent and remained unchanged during treatment with nifedipine. Treatment of GT1-7 neurons with the NO donor 3,3'-(hydroxynitrosohydrazino)bis-1-propanamine (DPTA) caused a massive increase in cGMP production that was significantly reduced by concomitant GnRH treatment. Inhibition of cGMP production by the non-selective NOS inhibitor, N(omega)-nitro-L-arginine-methylester (L-NAME), was dose-dependent and maximal inhibitory concentrations of L-NAME and GnRH were not additive, indicating NOS as a common site of action. Pretreatment of GT1-7 neurons with pertussis toxin (PTX) significantly attenuated the GnRH-induced reduction of cGMP production. Similarly, transfection of GT1-7 neurons with minigene vectors that produce Galpha C-terminal inhibitory peptides suppressed the catalytic activity of PTX-sensitive Gi and Galphao subunits, and prevented the inhibitory action of GnRH on cGMP production. However, blockade of the catalytic activities of Galphas and Galphaq by expression of specific C-terminal peptides did not affect the inhibitory action of GnRH on cGMP production. In summary, these data provide evidence that GnRH-R-activated PTX-sensitive G proteins inhibit NOS-sGC activity and reduce cGMP production in immortalized GnRH neurons.